Loss of cell cycle checkpoint control and acquisition of the ability to proliferate indefinitely constitute two of the fundamental changes required for the development of cancer. BRIT1 (BRCT-Repeat Inhibitor of hTERT expression), a novel gene identified through our enhanced retroviral mutation screen as a repressor of hTERT function, is implicated in cellular immortalization. Our preliminary data indicate that BRIT1 has additional functions-regulating both the intra-S and G2/M checkpoints-that, in part, may result from its regulation of BRCA1 and Chk1 expression and its direct role in the transmission of DNA damage signals. Moreover, BRIT1 is located on chromosome 8p23.1, which has been implicated in the development of several malignancies, including breast cancer and ovarian cancer, based on LOH and CGH analyses. The expression of BRIT1 also was significantly reduced in these two cancer types when analyzed by DNA arrays. Thus, we hypothesize that BRIT1 may function as a tumor suppressor gene through dual functions: repressing cellular immortalization and maintaining genomic stability. This hypothesis will be tested in the following steps. (1) We will determine the role of BR T1 in preventing cellular immortalization by using normal human cells that conditionally express small hairpin RNA against BRIT1, and we will investigate the mechanisms mediating BRIT1's function on hTERT expression. (2) We will use BRIT 1-depleted cells to study the role of BRIT1 in the response to genotoxic stress, maintenance of genomic integrity, and cellular transformation;we will assess genomic instability by detecting chromosome alterations using comparative genomic hybridization (CGH) and spectral karyotyping (SKY);we will study how BRIT1 regulates the promoter activities of BRCA1 and Chk1 and how BRIT1 may play a direct role in transmission of DNA damage signal;and we will develop a BRIT1 conditional knockout mouse model to assess the BRIT1's function in vivo in maintaining genomic integrity and suppressing tumor formation. (3) We will identify and characterize the aberrations of BRIT1 in breast and ovarian cancer samples to determine if BRIT1 serves as a prognostic marker or as a therapeutic target in cancer. As a corollary, an analysis of the function of BRIT1 and its aberrations in cancer patients will contribute to an improved understanding of the key pathological alterations in cancer initiation and progression and to the development of novel, effective therapeutic approaches for cancer.